Match ends, Czech Republic 2, Netherlands 1.
Guus Hiddink suffered his second straight defeat since taking charge of the Netherlands as Vaclav Pilar hit an injury-time winner for Czech Republic.
Borek Dockal had given the hosts the lead in the Euro 2016 qualifier in Prague before Stefan de Vrij equalised.
But Daryl Janmaat's mistake allowed Pilar to seal the points as Hiddink, 67, became the first Dutch national manager to lose his first two games since his previous reign in 1995.
Italy, Iceland and Cyprus also won.
Goals by Simone Zaza and Leonardo Bonucci earned the Azzurri a 2-0 win away to Norway to extend Antonio Conte's winning start since taking over following Cesare Prandelli's resignation.
Also in Group H, Real Madrid's former Tottenham midfielder Luka Modric and Andrej Kramaric scored as Croatia overcame 10-man Malta 2-0, while Bulgaria won 2-1 away to Azerbaijan.
One of the biggest surprises of the night saw Wales' Group B rivals Cyprus come from behind to win 2-1 away to Bosnia-Hercegovina.
Vedad Ibisevic put Bosnia ahead, but Demetris Christofi equalised and then scored again in the 73rd minute.
Bosnia's misery was complete when Roma midfielder Miralem Pjanic saw his penalty saved by Antonis Georgallides.
There was also a surprise in Group A where Iceland ran out 3-0 winners at home to 10-man Turkey.
Dadi Bodvarsson put them ahead before Turkey's Omer Toprak was sent off for two yellow cards.
Swansea's Gylfi Sigurdsson and Kolbeinn Sigthorsson scored two goals in one minute to round off the win.
In the same group, Kazakhstan drew 0-0 at home to Latvia.
- 5ProchazkaBooked at 8mins
- 8LimberskyBooked at 90mins
- 13VachaSubstituted forKolarat 81'minutes
- 7KrejciSubstituted forPilarat 66'minutes
- 21LafataSubstituted forVydraat 72'minutes
- 3VeltmanSubstituted forNarsinghat 39'minutes
- 4de Vrij
- 5Martins IndiBooked at 71mins
- 6de Jong
- 9van Persie
- 12Van der Wiel
- 13van Dijk
- Gianluca Rocchi
- Shots on Target
Second Half ends, Czech Republic 2, Netherlands 1.
Attempt missed. Robin van Persie (Netherlands) header from the centre of the box is close, but misses to the left. Assisted by Wesley Sneijder with a cross following a set piece situation.
David Limbersky (Czech Republic) is shown the yellow card for a bad foul.
Luciano Narsingh (Netherlands) wins a free kick on the right wing.
Foul by David Limbersky (Czech Republic).
Georginio Wijnaldum (Netherlands) wins a free kick in the attacking half.
Foul by Vaclav Pilar (Czech Republic).
Goal! Czech Republic 2, Netherlands 1. Vaclav Pilar (Czech Republic) right footed shot from very close range to the bottom right corner.
Offside, Netherlands. Nigel de Jong tries a through ball, but Memphis Depay is caught offside.
Corner, Czech Republic. Conceded by Robin van Persie.
Foul by Georginio Wijnaldum (Netherlands).
Vaclav Pilar (Czech Republic) wins a free kick in the attacking half.
Offside, Czech Republic. Petr Cech tries a through ball, but Matej Vydra is caught offside.
Hand ball by Georginio Wijnaldum (Netherlands).
Attempt missed. Vaclav Prochazka (Czech Republic) header from very close range is too high. Assisted by Pavel Kaderabek with a headed pass following a corner.
Substitution, Czech Republic. Daniel Kolár replaces Lukas Vacha.
Corner, Czech Republic. Conceded by Bruno Martins Indi.
Attempt saved. Memphis Depay (Netherlands) header from the centre of the box is saved in the centre of the goal. Assisted by Luciano Narsingh with a cross.
Memphis Depay (Netherlands) wins a free kick in the attacking half.